Structural Analysis of Treponema pallidum Lipoproteins

梅毒螺旋体脂蛋白的结构分析

• 批准号: 7147449
• 负责人: MICHAEL V. NORGARD
• 金额: $ 45.59万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2009-02-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147449
• 关键词: Address; Agonist; Arts; Bacteria; Chimeric Proteins; Complex; Data; Escherichia coli; Genes; Genome; Globus Pallidus; Human; Immune; Immune Targeting; Immune response; Investigation; Lipoproteins; Membrane; Membrane Biology; Membrane Proteins; Molecular; Order Spirochaetales; Pathogenesis; Physiological; Play; Process; Protein Conformation; Proteins; Research; Role; Sexually Transmitted Diseases; Structural Biologist; Structural Protein; Syphilis; Techniques; Treponema pallidum; Virulence Factors; X ray diffraction analysis; X-Ray Diffraction; novel; polypeptide; protein function; structural biology; success; three dimensional structure

Syphilis, caused by the spirochetal bacterium Treponema pallidum, continues to play prominently as a sexually transmitted disease. Syphilis pathogenesis also represents a paradigm of bacterial chronicity and immune evasion, but virtually nothing is known about how T. paltidum carries out these enigmatic processes. More specifically, the T. pallidum outer envelope, comprised of a cytoplasmic and outer membrane, must serve as both the physical and functional interface within the human host. Unfortunately, even though the T. pallidum genome has been sequenced, there remains a scarcity of information on the functions of T. pallidum membrane and membrane-associated proteins that likely contribute to the spirochete's complex parasitic strategy. Among the putative membrane proteins, T. pallidum is postulated to encode about 24-35 lipoproteins. Membrane lipoproteins of other bacteria subserve many important physiological roles and also have importance as virulence factors, modular components of ABC-type transporters, protective immune targets, and proinflammatory agonists that evoke robust innate immune responses. However, the functions of the treponemal lipoproteins remain essentially undefined. In a departure from more traditional approaches to T. pallidum research, the proposed study brings together a group of treponematologists, molecular biologists, protein biochemists, and structural biologists to address this important information gap in a novel way. The Specific Aims of this proposal are: (1) To clone and express in E. coli the lipoprotein genes of T. pallidum, with emphasis on expressing high quantities of each polypeptide as a nonacylated (soluble) fusion protein; (2) To purify to homogeneity each fusion protein and perform biophysical assessments of protein conformation; and (3) To obtain protein crystals suitable for X-ray diffraction and solve the three-dimensional structure for each crystallizable lipoprotein. Extensive preliminary data and progress in other structural biology initiatives support the timeliness and feasibility of this project; many state-of-the-art protein structural characterization techniques will enhance overall success. Finally, structural data will be used to formulate new testable hypotheses regarding potential function(s) of the lipoproteins, new avenues of investigation for T. pallidum membrane biology and syphilis pathogenesis that are sorely needed.

梅毒是由螺旋体细菌梅毒螺旋体引起的，作为一种常见的疾病，仍然发挥着重要作用。 性病。梅毒发病机制也代表了细菌慢性化和 免疫逃避，但实际上对 T. paltidum 如何进行这些神秘过程一无所知。 更具体地说，梅毒螺旋体外被膜由细胞质和外膜组成，必须 作为人类宿主内的物理和功能接口。不幸的是，尽管T. 梅毒螺旋体基因组已被测序，但有关梅毒螺旋体功能的信息仍然匮乏 膜和膜相关蛋白可能有助于螺旋体的复杂寄生 战略。在假定的膜蛋白中，梅毒螺旋体被假定编码约 24-35 个 脂蛋白。其他细菌的膜脂蛋白具有许多重要的生理作用，并且 作为毒力因子、ABC 型转运蛋白的模块化成分、保护性免疫具有重要意义 靶点和促炎激动剂，可引起强大的先天免疫反应。然而，功能 密螺旋体脂蛋白基本上仍然未定义。与更传统的方法不同 梅毒螺旋体研究，拟议的研究汇集了一群密螺旋体学家、分子生物学家、 蛋白质生物化学家和结构生物学家以一种新颖的方式解决这一重要的信息差距。这 该提案的具体目标是：（1）在大肠杆菌中克隆并表达梅毒螺旋体的脂蛋白基因， 重点是将每种多肽大量表达为非酰化（可溶性）融合蛋白； (2) 纯化每个融合蛋白至均质并对蛋白质构象进行生物物理评估； (3)获得适合X射线衍射的蛋白质晶体并解析其三维结构 每种可结晶脂蛋白。其他结构生物学计划的广泛初步数据和进展 支持该项目的及时性和可行性；许多最先进的蛋白质结构表征 技术将提高整体成功率。最后，结构数据将用于制定新的可测试的 关于脂蛋白潜在功能的假设，梅毒螺旋体研究的新途径 迫切需要膜生物学和梅毒发病机制。